Method for improving salidroside produced by genetically engineered bacteria and application of phosphoglucomutase mutant

Abstract

The invention discloses a method for improving salidroside produced by genetically engineered bacteria and application of a phosphoglucomutase mutant, and belongs to the technical field of bioengineering. The problem of low salidroside yield in the prior art is solved. The invention provides a phosphoglucomutase mutant, which is obtained by taking an amino acid sequence as shown in SEQ ID NO.2 as a starting sequence and mutating any one of a 5th site, a 114 site and a 431st site. The invention is mainly used for high-efficiency phosphoglucomutase and application of the phosphoglucomutase in de novo synthesis of salidroside.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to a method and application of a phosphoglucomutase mutant in improving the production of salidroside by genetically engineered bacteria. Background Art

[0002] Salidroside (CAS 10338-51-9) is a natural glycoside compound, chemically named 2-(4-hydroxyphenyl)ethyl- β -D-glucoside (molecular formula C 14 H 20 O7, molecular weight 300.3). Its physical and chemical properties include sweet taste and high water solubility (very soluble in water and methanol, poorly soluble in ether). Salidroside is the core active ingredient of the genus Rhodiola in the Rosaceae family, exhibiting multiple pharmacological activities, including anti-hypoxia, anti-fatigue, antioxidant, neuroprotective, and anti-tumor activities. With its expanding application in functional foods, cosmetics, and cardiovascular and cerebrovascular drugs, global market demand is growing by over 15% annually.

[0003] Extracting salidroside from natural plants faces significant bottlenecks. Wild Rhodiola rosea resources are scarce, with a growth cycle of 5-7 years and a mere 0.1-1% (dry weight), which is insufficient to meet market demand. While chemical synthesis allows for large-scale production, it faces challenges such as the use of toxic reagents (such as halogenated hydrocarbons and heavy metal catalysts), significant pollution, and a violation of green manufacturing principles. However, biosynthesis is considered an alternative due to its mild conditions, minimal byproducts, and environmental friendliness. Summary of the Invention

[0004] The purpose of the present invention is to increase the yield of salidroside.

[0005] The present invention provides a phosphoglucomutase mutant. The phosphoglucomutase mutant is obtained by taking the amino acid sequence shown in SEQ ID NO.2 as the starting sequence and mutating any one of the 5th, 114th and 431st positions.

[0006] The present invention provides a phosphoglucomutase mutant, wherein the phosphoglucomutase mutant is any one of the following: (1) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the asparagine at position 5 with proline; (2) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the asparagine at position 114 with aspartic acid; (3) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the glutamic acid at position 431 with glutamine.

[0007] The present invention provides a gene encoding the above-mentioned phosphoglucomutase mutant.

[0008] The present invention provides a recombinant vector containing the gene.

[0009] It is further defined that the starting vectors are pRSFDuet series and pETDuet series.

[0010] The present invention provides a recombinant microbial cell containing the gene.

[0011] Further defined, overexpression in Escherichia coli derived from Escherichia coli ( Escherichia coli ) phosphoglucomutase mutant gene pgm Mut and UDP-glucose pyrophosphorylase genes galU Derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene from Escherichia coli aroG fbr , derived from Zymomonas mobilis ( Zymomonas mobilis tyrC ) cyclohexadiene dehydrogenase gene Arabidopsis thaliana and from Arabidopsis thaliana ( AtUGT85A1 ) glycosyltransferase genes aroG The recombinant plasmid pETDuet- -tyrC-AtUGT85A1 fbr Escherichia coli .

[0012] The present invention provides a use of the above-mentioned phosphoglucomutase mutant, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the production of salidroside.

[0013] The present invention provides a method for producing salidroside, which comprises the following steps: fermenting the above-mentioned recombinant microbial cells at 30° C. for 72 hours.

[0014] Further limited, the fermentation medium ingredients are 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L monohydrate citric acid, 0.1 g / L vitamin B1, 7 g / L yeast powder, using 10 g / L CaCO3 to maintain the pH of the fermentation medium. Add 0.1% volume of trace elements to the medium, the trace element ingredients: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O, adjust the pH to 7.0 with NaOH.

[0015] Beneficial effect: Phosphoglucomutase plays a key role in activating the sugar donor in the biosynthesis of salidroside. Its role is to catalyze the formation of glucose-1-phosphate (G1P) from glucose-6-phosphate (G6P), which reacts under the catalysis of UTP-glucose-1-phosphate uridylyltransferase to generate UDP-glucose (UDPG). UDPG acts as a sugar donor, and the last step of salidroside biosynthesis is catalyzed by glycosyltransferase, which transfers the glucose group in UDPG to the C8 hydroxyl group of tyramine to form salidroside. If the supply of UDPG is insufficient, it will directly limit the synthesis efficiency of salidroside. Therefore, modifying phosphoglucomutase to obtain a high-efficiency phosphoglucomutase mutant is another effective way for high-efficiency biosynthesis of salidroside. DETAILED DESCRIPTION

[0016] The above fermentation medium ingredients for shake flask fermentation are 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L monohydrate citric acid, 0.1 g / L vitamin B1, 7 g / L yeast powder, 10 g / L CaCO3, 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, 15 g / L ZnSO4·7H2O, and the pH is adjusted to 7.0.

[0017] Example 1: Construction of genetically engineered bacteria containing different phosphoglucomutase mutants Overexpression from Escherichia coli ( pgm ) phosphoglucomutase gene galU and UDP-glucose pyrophosphorylase genes Saccharomyces cerevisiae Derived from Saccharomyces cerevisiae ( ARO10 phenylpyruvate decarboxylase gene ADH6 and alcohol dehydrogenase gene pgm-galU-ARO10-ADH6 The recombinant plasmid pRSFDuet- aroG and overexpression of a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene from Escherichia coli Zymomonas mobilis fbr , derived from Zymomonas mobilis ( tyrC ) cyclohexadiene dehydrogenase gene Arabidopsis thaliana and from Arabidopsis thaliana ( AtUGT85A1 ) glycosyltransferase genes aroG The recombinant plasmid pETDuet- -tyrC-AtUGT85A1 fbr Escherichia coli From the Engineering pgm forEfficient De Novo Synthesis of Salidroside).

[0018] The overexpression plasmid pRSFDuet- galU - ARO10 - ADH6 - pgm The wild-type phosphoglucomutase gene in pgm Replacement of phosphoglucomutase gene with different site-directed mutations pgm Mut (phosphoglucomutase gene pgm I3N , phosphoglucomutase gene pgm N5P , phosphoglucomutase gene pgm A33V , phosphoglucomutase gene pgm A36P , phosphoglucomutase gene pgm A52G , phosphoglucomutase gene pgm E113Q , phosphoglucomutase gene pgm N114D , phosphoglucomutase gene pgm F117Y , phosphoglucomutase gene pgmV123I phosphoglucomutase gene pgm V129E phosphoglucomutase gene pgm L137Q phosphoglucomutase gene pgm E195Q phosphoglucomutase gene pgm M197W phosphoglucomutase gene pgm M208I phosphoglucomutase gene pgm T230K phosphoglucomutase gene pgm P239S phosphoglucomutase gene pgm Y250F phosphoglucomutase gene pgm N252Q phosphoglucomutase gene pgm N254D phosphoglucomutase gene pgm E431Q phosphoglucomutase gene pgm N434D phosphoglucomutase gene pgm K438Q phosphoglucomutase gene pgm A451S ), 23 kinds of plasmids containing pgm mutants were obtained, and the above 23 kinds of plasmids were respectively combined with pETDuet- pgm fbr pgm The plasmid combination was introduced into the chassis strain Escherichia coli BL21 (DE3) to obtain 23 kinds of genetically engineered bacteria containing different phosphoglucomutase mutants. The strains were named by using the mutation amino acid sites.

[0019] Based on the amino acid sequence shown in SEQ ID NO. 2, the corresponding base substitution results are I3N, N5P, A33V, A36P, A52G, E113Q, N114D, F117Y, V123I, V129E, L137Q, E195Q, M197W, M208I, T230K, P239S, Y250F, N252Q, N254D, E431Q, N434D, K438Q and A451S, wherein the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid.

[0020] SEQ ID NO. 1 (nucleotide sequence of wild-type phosphoglucomutase pgm):

[0021] SEQ ID NO. 2 (nucleotide sequence of wild-type phosphoglucomutase pgm): MAIHNRAGQPAQQSDLINVAQLTAQYYVLKPEAGNAEHAVKFGTSGHRGSAARHSFNEPHILAIAQAIAEERAKNGITGPCYVGKDTHALSEPAFISVLEVLAANGVDVIVQENNGFTPTPAVSNAILVHNKKGGPLADGIVITPSHNPPEDGGIKYNPPNGGPADTNVTKVVEDRANALLADGLKGVKRISLDEAMASGHVKEQDLMQPFVEGLADIVDMAAIQKAGLTLGVDPLGGPGIEYWKRIGEYYNLNLTIVNDQVDQTFRFMHLDKDGAIRMDCSSECAMAGLLALRDKFDLAFANDPDYDRHGIVTPAGLMNPNHYLAVAINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKLVEVPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDKDGIIMCLLAAEITAVTGKNPQEHYNELAKRFGAPSYNRLQAAATSAQKAALSKLSPEMVSASTLAGDPITARLTAAPGNGASIGGLKVMTDNGWFAARPSGTEDAYKIYCESFLGEEHRKQIEKEAVEIVSEVLKNA*.

[0022] Table 1 genetically engineered bacteria containing different phosphoglucomutase mutants

[0023] Example 2: Rhodiolin fermentation experiment of genetically engineered bacteria containing different phosphoglucomutase mutants The 23 genetically engineered bacteria and the control strain (genotype of the strain is pgm BL21 (pRSFDuet- pgm pgm + pETDuet- pgm fbr pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pgm pg) inoculated into LB liquid medium containing resistance, the culture system was 100 mL triangular flask, 10 mL medium, the medium was supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin, the culture conditions were constant temperature shaking bed 37℃, 220 rpm overnight culture, seed liquid was obtained, the seed liquid was inoculated into fresh fermentation medium at a volume of 2%, the culture system was 250 mL triangular flask, 50 mL medium, the medium was supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin, the culture conditions were constant temperature shaking bed 37℃, 220 rpm, the culture OD 600 was 0.6-0.8, and 0.1 mM IPTG was added to induce, and the fermentation was induced at 30℃, 220 rpm for 72 h, and sample preparation was performed.

[0024] Example 3: Extraction and liquid phase detection of rhodioloside 1 mL of fermentation broth was taken into a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 5 min using a centrifuge, 200 μL of supernatant was mixed with 800 μL of mobile phase A, and the sample was diluted 5 times before being detected by a Shimadzu high performance liquid chromatograph to detect the yield of rhodioloside. The method was to use a reversed-phase C18 column for quantification. Mobile phase A was water containing 1% trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 1% TFA. The flow rate was 1 mL·min -1 , and the absorbance of the sample was detected at 274 nm. The HPLC program was as follows: 10% B: 0-5 min, 60% B: 5-10 min, 90% B: 10-12 min, 2% B: 12-16 min, 0% B: 16-20 min. The yield of rhodioloside in the shake flask fermentation of 23 different site-directed mutant phosphoglucomutases pgm mutants was statistically improved, and the results are shown in Table 2.

[0025] Table 2: Yield of rhodioloside in shake flask fermentation of different phosphoglucomutase pgm mutants

[0026] Note: The number + represents the improvement rate of rhodioloside yield; - represents no improvement.

[0027] Improvement rate (%) = (rhodioloside yield of mutant strain (g / L) - rhodioloside yield of control strain (g / L)) / rhodioloside yield of control strain (g / L) x 100%.

[0028] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the present application carries out site-directed mutagenesis on the phosphoglucomutase pgm derived from Escherichia coli, the phosphoglucomutase mutant pgm N5P The overexpression of the genetically engineered bacteria significantly improves the production of rhodioside, and the level of shake flask fermentation reaches 5.32 g / L, which is about 33% higher than that of the control strain, thereby providing valuable resources for constructing an efficient rhodioside production strain.

[0029] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A phosphoglucomutase mutant, characterized in that: The phosphoglucomutase mutant is obtained by taking the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence and mutating any one of the 5th, 114th and 431st positions.

2. A phosphoglucomutase mutant, characterized in that: The phosphoglucomutase mutant is any one of the following: (1) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the asparagine at position 5 with proline; (2) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the asparagine at position 114 with aspartic acid; (3) Using the amino acid sequence shown in SEQ ID NO. 2 as the starting sequence, replace the glutamic acid at position 431 with glutamine.

3. A gene encoding the phosphoglucomutase mutant according to claim 1 or 2.

4. A recombinant vector containing the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The starting vectors are pRSFDuet series and pETDuet series.

6. A recombinant microbial cell containing the gene according to claim 3.

7. The recombinant microbial cell according to claim 6, characterized in that Overexpression in Escherichia coli derived from Escherichia coli ( Escherichia coli ) phosphoglucomutase mutant gene pgm Mut and UDP-glucose pyrophosphorylase genes galU, Derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene from Escherichia coli aroG fbr , derived from Zymomonas mobilis ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and from Arabidopsis thaliana ( Arabidopsis thaliana ) glycosyltransferase genes AtUGT85A1 of the recombinant plasmid.

8. Use of the phosphoglucomutase mutant according to claim 1, the gene according to claim 3, the recombinant vector according to claim 4 or 5, or the recombinant microbial cell according to claim 6 or 7 in producing salidroside.

9. A method for producing salidroside, characterized in that: The method comprises the following steps: fermenting the recombinant microbial cells according to claim 7 at 30° C. for 72 hours.

10. The method according to claim 9, characterized in that The fermentation medium composition was 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast powder. 10 g / L CaCO3 was used to maintain the pH of the fermentation medium. The culture medium was supplemented with 0.1% volume of trace elements, including 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / LNiSO4·6H2O, and 15 g / L ZnSO4·7H2O, and the pH was adjusted to 7.0 using NaOH.

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